We have confirmed our preliminary observations that fewer hepatocytes of aged rats are capable of responding to epinephrine and EGF with increased DNA synthesis than corresponding cells of young animals. In addition the amount 3H thymidine incorporation per cell is also reduced. At the molecular level, we have obtained complimentary information to the age-related decrease in stimulated MAPK and increase in MAPK phosphatase activity reported last year. MEK1 and 2 stimulation is unaltered as are E2F levels, but stimulation of polymerase alpha and E2F binding to DNA are reduced with age. In addition, the MEK inhibitor PD098059 completely blocks stimulated DNA synthesis in young cells but does not affect old ones, suggesting that an alternative signal transduction pathway may be utilized in aged cells. For this reason, we are examining the PKC pathway which feeds into the MAPK pathway via ras, and have determined that inhibitors of PKC such as staurosporine and bisindolymaleimide block stimulated DNA synthesis in both young and old cells. Additionally, various isoforms of PKC have been identified. We have also determined that stimulation of the replication of specific genes, including myc and dihydrofolate reductase are also reduced with age, although there is no change in the replication of mitochondrial DNA which utilizes polymerase gamma rather than alpha and is cell cycle independent. 2) G protein mediated signal transduction and aging. We continue to test the hypothesis that the coupling/uncoupling of G proteins and receptors is altered during aging as a consequence of changes in plasma membrane composition. Although much of our initial work utilized isolated parotid cell aggregates, our current studies, which are largely directed toward development of methodology, employ striatal membrane preparations, which we have also studied extensively (see Z01-AG00306-7 LCMB).